A. Cell Death-Related Disorders
The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.
Anomalous cell death is a component of a number of pathologies, including neurodegenerative disease, cerebral amyloid beta-protein angiopathy (CAA), and some cardiomyopathies. Said anomalous cell death may comprise apoptotic cell death. Agents that are cell death-protective have utility for the prevention or treatment of said pathologies. In particular, agents that are neuroprotective or myoprotective have utility for the prevention or treatment of said neurodegenerative disease or said myopathy.
Alzheimer's disease is a progressive neurodegenerative disease characterized by significant loss of function in more than one cognitive domain and often accompanied by changes in behavior or personality (Alzheimer's Disease; Decision Resources, Inc.; November 2001). Mutations in three different genes can cause early-onset familial Alzheimer's disease (FAD): amyloid precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2). Early-onset familial Alzheimer's disease accounts for less than 5% of all Alzheimer's disease cases, however. More typically, Alzheimer's disease occurs in people aged 65 and older. Alzheimer's disease is the most common cause of dementia, affecting approximately 20 million people worldwide. Amyloid beta-peptide (Aβ) is believed to play a central role in the pathology of Alzheimer's disease.
Mild cognitive impairment (MCI) is a state of cognitive impairment, most often of memory only, that is not severe enough to significantly impact occupational or social functioning. However, many people with MCI progress to Alzheimer's disease, at a rate of 10-15% per year and up to 60% within five years. Growing evidence shows that Alzheimer's disease pathology begins much earlier than the development of symptoms severe enough to allow a diagnosis of Alzheimer's criteria. In 2001, the American Academy of Neurology (AAN) published clinical practice parameters for the detection of MCI that will increase awareness, diagnosis, and treatment of both MCI and mild forms of Alzheimer's disease.
Parkinson's disease is a chronic, progressive neurodegenerative disorder affecting more than 2.7 million people in the United States, Japan, and Western Europe. Parkinson's disease is characterized by motor symptoms such as tremor, bradykinesia, muscle rigidity, gait dysfunction, and postural instability. Although researchers have identified degeneration of dopaminergic neurons in the substantia nigra as the primary pathophysiological mechanism, they believe that other neurotransmitter systems—such as the serontonergic and glutamatergic systems—are also intricately involved in the disease process.
Prion-associated diseases include, but are not held to be limited to, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia [Collins et al., Lancet (2004) 3:51-61].
Stroke is an acute interruption of blood flow in the brain caused by blood vessel obstruction or hemorrhage. Almost 90% of all strokes are ischemic; that is, they are due to a reduction in cerebral circulation below a critical threshold. Stroke accounts for 10-12% of medically related deaths in industrialized countries and is the third leading cause of death worldwide. Overall estimates of the annual number of strokes in the United States range from 500,000 to 700,000.
Motor neuron disease is characterized by a selective degeneration of the motor neurons of the spinal cord, brainstem, or motor cortex. Clinical subtypes are distinguished by the major site of degeneration. In amyotrophic lateral sclerosis there is involvement of upper, lower, and brainstem motor neurons. In progressive spinal muscular atrophy and related syndromes the motor neurons in the spinal cord are primarily affected. With progressive bulbar palsy, the initial degeneration occurs in the brainstem. In primary lateral sclerosis, the cortical neurons are affected in isolation.
Peripheral neuropathy is a broad term used to describe a variety of disorders which manifest as painful or uncomfortable sensations usually in the extremities and are of neuropathic origin. Nerve degeneration is believed to play a role in peripheral neuropathy. This condition is found commonly amongst end stage renal disease and diabetic patients. Distal symmetrical polyneuropathy (DSP)—a neuropathy affecting both sides of the body and attacking the distal sensory nerves—is the most common clinical manifestation of diabetic neuropathy and accounts for approximately 80-85% of all neuropathy observed in diabetic patients. DSP affects 30-40% of patients with type 1 and type 2 diabetes. Diabetic neuropathy can typically damage many different nerves, including the sciatic nerve.
Cerebral amyloid beta-protein (Aβ) angiopathy (CAA) is a key feature of Alzheimer's disease and related disorders. CAA is a disease of small blood vessels in the brain in which deposits of amyloid protein in the vessel walls may lead to stroke, brain hemorrhage, or dementia. Vascular amyloid deposition leads to degeneration of the vessel wall and aneurysm formation, and may be responsible for 10 to 15% of hemorrhagic strokes in the elderly.
Myocardial infarction is the damage or death of an area of heart muscle because of an inadequate supply of oxygen to that area. Myocardial infarctions are often caused by a clot that blocks one of the coronary arteries (the blood vessels that bring blood and oxygen to heart muscle). The clot prevents blood and oxygen from reaching that area of the heart, leading to the death of cardiomyocytes in that area.
Congestive heart failure affects nearly 5 million Americans with over 500,000 new cases diagnosed annually. Ischemic heart disease is the most common cause of congestive heart failure, accounting for 60-70% of all cases. By definition, congestive heart failure is a clinical syndrome in which heart disease reduces cardiac output, increases venous pressures, and is accompanied by molecular abnormalities that cause progressive deterioration of the failing heart and premature cardiomyocyte (cardiac muscle cell) death. Any definition of heart failure that does not consider the molecular processes that accelerate myocardial death overlooks a major clinical feature of this syndrome. Evidence in mice and rats demonstrate that inhibitors of cardiomyocyte death pathways significantly improve cardiac function and animal survival [Laugwitz et al., Hum Gene Ther (2001) 12:2051-63].
B. Humanin
The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.
Humanin polypeptide was originally identified through its capacity to protect neuronal cells from Alzheimer's disease-relevant toxicity [Hashimoto et al., Biochem Biophys Res Commun (2001) 283:460-8]. Humanin has the amino acid sequence: Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala (SEQ ID NO. 15) (GenBank® Accession No. AAK50430). An analog of Humanin having a substitution of glycine for serine at amino acid position 14, [Gly14]-Humanin, has been reported to be significantly more active than Humanin [Niikura et al., J Neurosci Res (2002) 70:380-91]. Humanin immunoreactivity has been detected in an Alzheimer's disease brain, but little in normal human brains [Tajima et al., Neurosci Lett (2002) 324:227-31].
Humanin has been found to protect neuronal cells from a number of toxic insults. This includes neurotoxicity mediated by three mutant genes that cause FAD as well as by Aβ [Hashimoto et al., Proc Natl Acad Sci USA (2001) 98:6336-41; Niikura et al., J Neurosci Res (2002) 70:380-91]. Aβ neurotoxicity involves a c-Jun N-terminal kinase (JNK) pathway, and Humanin acts at a point downstream of JNK [Hashimoto et al., J Neurochem (2003) 84:864-77]. Humanin has also been reported to have protective activity for neurons against serum deprivation [Takahashi et al., Neuroreport (2002) 13:903-7]. The rat ortholog of Humanin has been reported to have protective activity for neurons against excitotoxic death [Caricasole et al., FASEB J (2002) 16:1331-3].
Humanin has also been shown to rescue cortical neurons from prion-peptide-induced apoptosis [Sponne et al., Mol Cell Neurosci (2004) 25:95-102].
Humanin has further been shown to improve learning and memory impairment in mice, thereby evidencing utility as a beneficial agent for the prevention or treatment of said learning or memory impairment [Mamiya & Ukai, Br J Pharmacol (2001) 134:1597-9].
Recently, Humanin has been shown to be protective for muscle cells. Humanin rescues human cerebrovascular smooth muscle cells from Aβ-induced toxicity.
Collectively, the foregoing indicates that Humanin and compounds evidencing Humanin activity are cell death-protective. Said cell death may comprise apoptotic cell death. In particular, Humanin and compounds evidencing Humanin activity protect neuronal cells and muscle cells from cell death and have utility for the prevention or treatment of neurodegenerative disease, cerebral amyloid angiopathy, and some cardiomyopathies.
C. G Protein-Coupled Receptors
The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR) class. It is estimated that there are some 30,000-40,000 genes within the human genome, and of these, approximately 2% are estimated to code for GPCRs.
GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription pharmaceuticals have been developed. For example, in 1999, of the top 100 brand name prescription drugs, the following drugs interact with GPCRs (the primary diseases and/or disorders treated related to the drug is indicated in parentheses):
Claritin ® (allergies)Prozac ® (depression)Vasotec ® (hypertension)Paxil ® (depression)Zoloft ® (depression)Zyprexa ®(psychotic disorder)Cozaar ® (hypertension)Imitrex ® (migraine)Zantac ® (reflux)Propulsid ® (reflux disease)Risperdal ® (schizophrenia)Serevent ® (asthma)Pepcid ® (reflux)Gaster ® (ulcers)Atrovent ® (bronchospasm)Effexor ® (depression)Depakote ® (epilepsy)Cardura ®(prostatic ypertrophy)Allegra ® (allergies)Lupron ® (prostate cancer)Zoladex ® (prostate cancer)Diprivan ® (anesthesia)BuSpar ® (anxiety)Ventolin ® (bronchospasm)Hytrin ® (hypertension)Wellbutrin ® (depression)Zyrtec ® (rhinitis)Plavix ® (MI/stroke)Toprol-XL ® (hypertension)Tenormin ® (angina)Xalatan ® (glaucoma)Singulair ® (asthma)Diovan ® (hypertension)Harnal ® (prostatic hyperplasia)(Med Ad News 1999 Data).
GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
Generally, when a ligand binds with the receptor (often referred to as “activation” of the receptor), there is a change in the conformation of the receptor that facilitates coupling between the intracellular region and an intracellular “G-protein.” It has been reported that GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Although not wishing to be bound to theory, it is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
There are also promiscuous G proteins, which appear to couple several classes of GPCRs to the phospholipase C pathway, such as Gα15 or Gα16 [Offermanns & Simon, J Biol Chem (1995) 270:15175-80], or chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C [Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20:118-24].
Gi-coupled GPCRs lower intracellular cAMP levels. A Gi-like G protein is a G protein that similarly leads to lower intracellular cAMP level. By this criterion, Go and Gz are Gi-like G proteins. The melanophore technology (see infra) is useful for identifying Gi-coupled GPCRs and also for identifying modulators of said Gi-coupled GPCRs.
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by a ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor. Stabilization by such ligand-independent means is termed “constitutive receptor activation.”
D. FPRL2
The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.
FPRL2 (GenBank® Accession No. L14061) is a GPCR for which no endogenous ligand has yet been demonstrated. The synthetic peptide WKYMVm (SEQ ID NO: 16) recently has been shown to bind to FPRL2 on mature human dendritic cells and elicit an increase in intracellular Ca2+ [Yang et al., J Leukoc Biol (2002) 72:598-607].
FPRL2 is most similar to the GPCRs FPRL1 (GenBank® Accession No. AF054013) and FPR (GenBank® Accession No. M60627). Recently, it was reported that Humanin uses FPRL1 as a functional receptor [Ying et al., Journal of Interferon and Cytokine Research (2002) 22 (Supplement 1):S180]. No endogenous ligand for FPRL1 or FPR has been shown to also be a ligand for FPRL2 and, in fact, a number of endogenous ligands for FPRL1 or FPR (lipoxin A4, e.g.) have been shown not to be ligands for FPRL2.